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Monday, 18 August 2014

Researchers have long suspected that major mental disorders are genetically-rooted diseases of synapses – the connections between neurons. Now, investigators supported in part by the National Institutes of Health have demonstrated in patients' cells how a rare mutation in a suspect gene disrupts the turning on and off of dozens of other genes underlying these connections.

Synapses – sites of intercellular communications

– are revealed in a mature iPSC cortex neuron

derived from a participant in the study. Immune-

based staining shows synapse markers (red,

green) and the cell's nucleus (blue). Credit:

Hongjun Song, Ph.D., Johns Hopkins University.

"Our results illustrate how genetic risk, abnormal brain development and synapse dysfunction can corrupt brain circuitry at the cellular level in complex psychiatric disorders," explained Hongjun Song, Ph.D., of Johns Hopkins University, Baltimore, a grantee of the NIH's National Institute of Mental Health (NIMH), a founder of the study.

Song and colleagues, from universities in the United States, China, and Japan, report on their discovery in the journal Nature, August 17, 2014.

"The approach used in this study serves as a model for linking genetic clues to brain development," said NIMH director Thomas R. Insel, M.D..

Most major mental disorders, such as schizophrenia, are thought to be caused by a complex interplay of multiple genes and environmental factors. However, studying rare cases of a single disease-linked gene that runs in a family can provide shortcuts to discovery. Decades ago, researchers traced a high prevalence of schizophrenia and other major mental disorders – which often overlap genetically – in a Scottish clan to mutations in the gene DISC1 (Disrupted In Schizophrenia-1). But until now, most of what's known about cellular effects of such DISC1 mutations has come from studies in the rodent brain.

To learn how human neurons are affected, Song's team used a disease-in-a-dish technology called induced pluripotent stem cells (iPSCs). A patient's skin cells are first induced to revert to stem cells. Stem cells play a critical role in development of the organism by transforming into the entire range of specialized cells which make up an adult. In this experiment, these particular "reverted" stem cells were coaxed to differentiate into neurons, which could be studied developing and interacting in a petri dish. This makes it possible to pinpoint, for example, how a particular patient's mutation might impair synapses. Song and colleagues studied iPSCs from four members of an American family affected by DISC1-linked schizophrenia and genetically related mental disorders.

Strikingly, iPSC-induced neurons, of a type found in front brain areas implicated in psychosis, expressed 80 percent less of the protein made by the DISC1 gene in family members with the mutation, compared to members without the mutation. These mutant neurons showed deficient cellular machinery for communicating with other neurons at synapses.

The researchers traced these deficits to errant expression of genes known to be involved in synaptic transmission, brain development, and key extensions of neurons where synapses are located. Among these abnormally expressed genes were 89 previously linked to schizophrenia, bipolar disorder, depression, and other major mental disorders. This was surprising, as DISC1's role as a hub that regulates expression of many genes implicated in mental disorders had not previously been appreciated, say the researchers.

The clincher came when researchers experimentally produced the synapse deficits by genetically engineering the DISC1 mutation into otherwise normal iPSC neurons – and, conversely, corrected the synapse deficits in DISC1 mutant iPSC neurons by genetically engineering a fully functional DISC1 gene into them. This established that the DISC1 mutation, was, indeed the cause of the deficits.

The results suggest a common disease mechanism in major mental illnesses that integrates genetic risk, aberrant neurodevelopment, and synapse dysfunction. The overall approach may hold promise for testing potential treatments to correct synaptic deficits, say the researchers.

A genetic variation linked to schizophrenia, bipolar disorder and severe depression wreaks havoc on connections among neurons in the developing brain, a team of researchers reports. The study, led by Guo-li Ming, M.D., Ph.D., and Hongjun Song, Ph.D., of the Johns Hopkins University School of Medicine and described online Aug. 17 in the journal Nature, used stem cells generated from people with and without mental illness to observe the effects of a rare and pernicious genetic variation on young brain cells. The results add to evidence that several major mental illnesses have common roots in faulty "wiring" during early brain development.

In this
image, cell nuclei are shown in blue and

synapses
in red and green. Credit: Zhexing Wen-

Johns Hopkins Medicine.

"This was the next best thing to going back in time to see what happened while a person was in the womb to later cause mental illness," says Ming.

"We found the most convincing evidence yet that the answer lies in the synapses that connect brain cells to one another."

Previous evidence for the relationship came from autopsies and from studies suggesting that some genetic variants that affect synapses also increase the chance of mental illness. But those studies could not show a direct cause-and-effect relationship, Ming says.

One difficulty in studying the genetics of common mental illnesses is that they are generally caused by environmental factors in combination with multiple gene variants, any one of which usually could not by itself cause disease. A rare exception is the gene known as disrupted in schizophrenia 1 (DISC1), in which some mutations have a strong effect. Two families have been found in which many members with the DISC1 mutations have mental illness.

Video
of human neurons firing. Credit: Zhexing

Wen-Johns Hopkins Medicine.

To find out how a DISC1 variation with a few deleted DNA "letters" affects the developing brain, the research team collected skin cells from a mother and daughter in one of these families who have neither the variation nor mental illness, as well as the father, who has the variation and severe depression, and another daughter, who carries the variation and has schizophrenia. For comparison, they also collected samples from an unrelated healthy person. Postdoctoral fellow Zhexing Wen, Ph.D., coaxed the skin cells to form five lines of stem cells and to mature into very pure populations of synapse-forming neurons.

After growing the neurons in a dish for six weeks, collaborators at Pennsylvania State University measured their electrical activity and found that neurons with the DISC1 variation had about half the number of synapses as those without the variation. To make sure that the differences were really due to the DISC1 variation and not to other genetic differences, graduate student Ha Nam Nguyen spent two years making targeted genetic changes to three of the stem cell lines.

In one of the cell lines with the variation, he swapped out the DISC1 gene for a healthy version. He also inserted the disease-causing variation into one healthy cell line from a family member, as well as the cell line from the unrelated control. Sure enough, the researchers report, the cells without the variation now grew the normal amount of synapses, while those with the inserted mutation had half as many.

"We had our definitive answer to whether this DISC1 variation is responsible for the reduced synapse growth," Ming says.

To find out how DISC1 acts on synapses, the researchers also compared the activity levels of genes in the healthy neurons to those with the variation. To their surprise, the activities of more than 100 genes were different.

"This is the first indication that DISC1 regulates the activity of a large number of genes, many of which are related to synapses," Ming says.

The research team is now looking more closely at other genes that are linked to mental disorders. By better understanding the roots of mental illness, they hope to eventually develop better treatments for it, Ming says.

Wednesday, 13 August 2014

Gene-based personalized medicine has many possibilities for diagnosis and targeted therapy, but one big bottleneck: the expensive and time-consuming DNA-sequencing process.

A DNA molecule passes through a nanopore in a

sheet of molybdenum disulphide, a material that

researchers have found to be better than

graphene at reading the DNA sequence.

Credit: Narayana Aluru, University of Illinois.

Now, researchers at the University of Illinois at Urbana-Champaign have found that nanopores in the material molybdenum disulphide (MoS2) could sequence DNA more accurately, quickly and inexpensively than anything yet available.

"One of the big areas in science is to sequence the human genome for under $1,000, the 'genome-at-home,'" said Narayana Aluru, a professor of mechanical science and engineering at the U. of I. who led the study.

"There is now a hunt to find the right material. We've used MoS2 for other problems, and we thought, why don't we try it and see how it does for DNA sequencing?"

As it turns out, MoS2 outperforms all other materials used for nanopore DNA sequencing – even graphene.

A nanopore is a very tiny hole drilled through a thin sheet of material. The pore is just big enough for a DNA molecule to thread through. An electric current drives the DNA through the nanopore, and the fluctuations in the current as the DNA passes through the pore tell the sequence of the DNA, since each of the four letters of the DNA alphabet – A, C, G and T – are slightly different in shape and size.

Illinois researchers found that the material

molybdenum disulphide could be the most

efficient yet found for DNA sequencing, making

personalized medicine more accessible. From

left: Amir Barati Farimani, Kyongmin Min and

Narayana Aluru. Credit: L. Brian Stauffer.

Most materials used for nanopore DNA sequencing have a sizable flaw: They are too thick. Even a thin sheet of most materials spans multiple links of the DNA chain, making it impossible to accurately determine the exact DNA sequence.

Graphene has become a popular alternative, since it is a sheet made of a single layer of carbon atoms – meaning only one base at a time goes through the nanopore. Unfortunately, graphene has its own set of problems, the biggest being that the DNA sticks to it. The DNA interacting with the graphene introduces a lot of noise that makes it hard to read the current, like a radio station marred by loud static.

MoS2 is also a single-layer sheet, thin enough that only one DNA letter at a time goes through the nanopore. In the study, the Illinois researchers found that DNA does not stick to MoS2, but threads through the pore cleanly and quickly. See an animation online.

"MoS2 is a competitor of graphene in terms of transistors, but we showed here a new functionality of this material by showing that it is capable of biosensing," said graduate student Amir Barati Farimani, the first author of the paper.

Most exciting for the researchers, the simulations yielded four distinct signals corresponding to the bases in a double-stranded DNA molecule. Other systems have yielded two at best – A/T and C/G – which then require extensive computational analysis to attempt to distinguish A from T and C from G.

The key to the success of the complex MoS2 simulation and analysis was the Blue Waters supercomputer, located at the National Center for Supercomputing Applications at the U. of Illinois.

"These are very detailed calculations," said Aluru, who is also a part of the Beckman Institute for Advanced Science and Technology at the U. of I.

"They really tell us the physics of the actual mechanisms, and why MoS2 is performing better than other materials. We have those insights now because of this work, which used Blue Waters extensively."

Now, the researchers are exploring whether they can achieve even greater performance by coupling MoS2 with another material to form a low-cost, fast and accurate DNA sequencing device.

"The ultimate goal of this research is to make some kind of home-based or personal DNA sequencing device," Barati Farimani said.

"We are on the path to get there, by finding the technologies that can quickly, cheaply and accurately identify the human genome. Having a map of your DNA can help to prevent or detect diseases in the earliest stages of development. If everybody can cheaply sequence so they can know the map of their genetics, they can be much more alert to what goes on in their bodies."

Bioengineers at the University of California, San Diego have proven that when it comes to guiding stem cells into a specific cell type, the stiffness of the extracellular matrix used to culture them really does matter. When placed in a dish of a very stiff material, or hydrogel, most stem cells become bone-like cells. By comparison, soft materials tend to steer stem cells into soft tissues such as neurons and fat cells. The research team, led by bioengineering professor Adam Engler, also found that a protein binding the stem cell to the hydrogel is not a factor in the differentiation of the stem cell as previously suggested. The protein layer is merely an adhesive, the team reported Aug. 10 in the advance online edition of the journal Nature Materials.

Their findings affirm Engler's prior work on the relationship between matrix stiffness and stem cell differentiations.

Cells grown on three hydrogels of the same

stiffness all display fat cell markers and
deform

the underlying matrix material in the same way.

Credit: Adam Engler, UC San Diego Jacobs

School of Engineering.

"What's remarkable is that you can see that the cells have made the first decisions to become bone cells, with just this one cue. That's why this is important for tissue engineering," said Engler, a professor at the UC San Diego Jacobs School of Engineering.

Engler's team, which includes bioengineering graduate student researchers Ludovic Vincent and Jessica Wen, found that the stem cell differentiation is a response to the mechanical deformation of the hydrogel from the force exerted by the cell. In a series of experiments, the team found that this happens whether the protein tethering the cell to the matrix is tight, loose or non-existent. To illustrate the concept, Vincent described the pores in the matrix as holes in a sponge covered with ropes of protein fibres. Imagine that a rope is draped over a number of these holes, tethered loosely with only a few anchors or tightly with many anchors. Across multiple samples using a stiff matrix, while varying the degree of tethering, the researchers found no difference in the rate at which stem cells showed signs of turning into bone-like cells. The team also found that the size of the pores in the matrix also had no effect on the differentiation of the stem cells as long as the stiffness of the hydrogel remained the same.

Cells grown on three hydrogels of the same

stiffness all display fat cell markers and
deform

the underlying matrix material in the same way.

Credit: Adam Engler/UC San Diego Jacobs

School of Engineering.

"We made the stiffness the same and changed how the protein is presented to the cells (by varying the size of the pores and tethering) and ask whether or not the cells change their behaviour," Vincent said.

"Do they respond only to the stiffness? Neither the tethering nor the pore size changed the cells."

"We're only giving them one cue out of dozens that are important in stem cell differentiation," said Engler.

"That doesn't mean the other cues are irrelevant; they may still push the cells into a specific cell type. We have just ruled out porosity and tethering, and further emphasized stiffness in this process."

The therapy was found to be safe, and all the patients showed improvements in clinical measures of disability.

The findings are published in the journal Stem Cells Translational Medicine. It is the first UK human trial of a stem cell treatment for acute stroke to be published.

The therapy uses a type of cell called CD34+ cells, a set of stem cells in the bone marrow that give rise to blood cells and blood vessel lining cells. Previous research has shown that treatment using these cells can significantly improve recovery from stroke in animals. Rather than developing into brain cells themselves, the cells are thought to release chemicals that trigger the growth of new brain tissue and new blood vessels in the area damaged by stroke.

The patients were treated within seven days of a severe stroke, in contrast to several other stem cell trials, most of which have treated patients after six months or later. The Imperial researchers believe early treatment may improve the chances of a better recovery.

A bone marrow sample was taken from each patient. The CD34+ cells were isolated from the sample and then infused into an artery that supplies the brain. No previous trial has selectively used CD34+ cells, so early after the stroke, until now.

Although the trial was mainly designed to assess the safety and tolerability of the treatment, the patients all showed improvements in their condition in clinical tests over a six-month follow-up period.

Four out of five patients had the most severe type of stroke: only four per cent of people who experience this kind of stroke are expected to be alive and independent six months later. In the trial, all four of these patients were alive and three were independent after six months.

Dr Soma Banerjee, a lead author and Consultant in Stroke Medicine at Imperial College Healthcare NHS Trust, said:

"This study showed that the treatment appears to be safe and that it's feasible to treat patients early when they might be more likely to benefit. The improvements we saw in these patients are very encouraging, but it's too early to draw definitive conclusions about the effectiveness of the therapy. We need to do more tests to work out the best dose and timescale for treatment before starting larger trials."

Over 150,000 people have a stroke in England every year. Survivors can be affected by a wide range of mental and physical symptoms, and many never recover their independence.

Stem cell therapy is seen as an exciting new potential avenue of treatment for stroke, but its exact role is yet to be clearly defined.

Dr Paul Bentley, also a lead author of the study, from the Department of Medicine at Imperial College London, said:

"This is the first trial to isolate stem cells from human bone marrow and inject them directly into the damaged brain area using keyhole techniques. Our group are currently looking at new brain scanning techniques to monitor the effects of cells once they have been injected."

Professor Nagy Habib, Principal Investigator of the study, from the Department of Surgery and Cancer at Imperial College London, said:

"These are early but exciting data worth pursuing. Scientific evidence from our lab further supports the clinical findings and our aim is to develop a drug, based on the factors secreted by stem cells, which could be stored in the hospital pharmacy so that it is administered to the patient immediately following the diagnosis of stroke in the emergency room. This may diminish the minimum time to therapy and therefore optimise outcome. Now the hard work starts to raise funds for this exciting research."

Tuesday, 5 August 2014

Knowing what to keep and what to trash: how an enzyme distinguishes cellular messages

Tuesday, 05 August 2014

Every once in a while, we are forced to sort that stack of papers on the kitchen counter. Interspersed between the expired coupons and dozens of takeout menus are important documents like your car insurance or electric bill. So it is not an option to simply drop it all in the trash at once – you need to read through the messages to be sure that you don’t lose vital information.

Dis3l2 is protein that preserves the character
of

stem cells by degrading specific messages. But

how does the enzyme know which messages to

destroy? CSHL researchers obtained a molecular

photograph (imaged on the left) of the protein
in

complex with a “poly-U” chain (in orange) that

marks messages for degradation. The poly-U

chain is inserted into a funnel-like structure
deep

within Dis3l2 (see cartoon on the right, poly-U

chain again in orange). Inside the funnel,
there

are more than a dozen contacts that capture the

poly-U mark so that the entire message can be

destroyed. Credit: Leemor Joshua-Tor,
Cold

Spring Harbor Laboratory.

In the cell, proteins similarly read through messages to distinguish what needs to be saved and what needs to be discarded. But, here, the process takes on a much more important role. More than just clutter, messages that are marked for disposal can drastically alter the fate of a cell. In fact, stem cells use just such a mechanism to maintain their identity. So how does a protein detect the difference between two seemingly similar messages? Today, a team of Cold Spring Harbor Laboratory (CSHL) scientists, led by Professor and Howard Hughes Medical Institute Investigator Leemor Joshua-Tor, describe how the protein Dis3l2 uses numerous recognition sites to capture messages that are flagged for decay.

Dis3l2 is a molecular machine that helps to preserve the character of stem cells. It serves as the executioner of an elegant pathway that prevents stem cells from changing into other cell types. The protein does this by acting like a garbage disposal for messages in the cell, cutting them up until they no longer encode useful information. But Dis3l2 is necessarily highly specific. While it must degrade messages that would alter the fate of the stem cell, discarding the wrong message could have devastating consequences.

Therefore, Dis3l2 only targets specific messages that have been marked with a molecular flag, known as a “poly-U” chain. The enzyme ignores the majority of messages in the cell – those that go on to encode proteins and other critical messages – whose ends are decorated with a different type of chain, called “poly-A” tail.

The CSHL scientists set out to understand how Dis3l2 is able to read and distinguish between these two chains. In work published today in Nature, they used a type of molecular photography, known as X-ray crystallography, to observe the structure of Dis3l2 while bound to a poly-U chain.

“We saw that the enzyme looks a lot like funnel, quite wide at the top and narrow at the base,” says Joshua-Tor.

“The poly-U chain inserts itself into the depths of this funnel while the rest of the bulky message can remain in the wide mouth at the top.”

But how does the enzyme “read” the poly-U chain? Christopher Faehnle, PhD and Jack Walleshauser, lead authors on the paper, found that the interior of the funnel contains more than a dozen contacts that interact specifically with the poly-U chain.

“Together, all of these points create a sticky web that holds the poly-U sequence deep within the enzyme,” says Faehnle.

“But other chains don’t interact – they can slide right out. It has helped us understand how an enzyme can differentiate between two sequences in the cell.”

More than that, the work provides insight into how a stem cell maintains its identity.

“Misregulation of any step in this pathway leads to developmental disorders and cancer,” says Joshua-Tor.

“We now have a much better appreciation of the terminal step, a critical point of control.”